The work presented in this thesis applies integrative genomic and experimental approaches to investigate mechanisms involved in regulation of gene expression in the context of disease and normal cell biology. In papers I and II, we have explored the role of epigenetic regulation of gene expression in multiple myeloma (MM). By using a bioinformatic approach we identified the Polycomb repressive complex 2 (PRC2) to be a common denominator for the underexpressed gene signature in MM. By using inhibitors of the PRC2 we showed an activation of the genes silenced by H3K27me3 and a reduction in the tumor load and increased overall survival in the in vivo 5TMM model. Using ChIP-sequencing we defined the distribution of H3K27me3 and H3K4me3 marks in MM patients cells. In an integrated bioinformatic approach, the H3K27me3-associated genes significantly correlated to under-expression in patients with less favorable survival. Thus, our data indicates the presence of a common under-expressed gene profile and provides a rationale for implementing new therapies focusing on epigenetic alterations in MM. In paper III we address the existence of a small cell population in MM presenting with differential tumorigenic properties in the 5T33MM murine model. We report that the predominant population of CD138+ cells had higher engraftment potential, higher clonogenic growth, whereas the CD138- MM cells presented with less mature phenotype and higher drug resistance. Our findings suggest that while designing treatment regimes for MM, both the cellpopulations must be targeted. In paper IV we have studied the general mechanism of differential gene expression regulation by CGGBP1 in response to growth signals in normal human fibroblasts. We found that CGGBP1 binding affects global gene expression by RNA Polymerase II. This is mediated by Alu RNAdependentinhibition of RNA Polymerase II. In presence of growth signals CGGBP1 is retained in the nuclei and exhibits enhanced Alu binding thus inhibiting RNA Polymerase III binding on Alus. Hence we suggest a mechanism by which CGGBP1 orchestrates Alu RNA-mediated regulation of RNA Polymerase II. This thesis provides new insights for using integrative bioinformatic approaches to decipher gene expression regulation mechanisms in MM and in normal cells.

The work presented in this thesis applies integrative genomic and experimental approaches to investigate mechanisms involved in regulation of gene expression in the context of disease and normal cell biology. In papers I and II, we have explored the role of epigenetic regulation of gene expression in multiple myeloma (MM). By using a bioinformatic approach we identified the Polycomb repressive complex 2 (PRC2) to be a common denominator for the underexpressed gene signature in MM. By using inhibitors of the PRC2 we showed an activation of the genes silenced by H3K27me3 and a reduction in the tumor load and increased overall survival in the in vivo 5TMM model. Using ChIP-sequencing we defined the distribution of H3K27me3 and H3K4me3 marks in MM patients cells. In an integrated bioinformatic approach, the H3K27me3-associated genes significantly correlated to under-expression in patients with less favorable survival. Thus, our data indicates the presence of a common under-expressed gene profile and provides a rationale for implementing new therapies focusing on epigenetic alterations in MM. In paper III we address the existence of a small cell population in MM presenting with differential tumorigenic properties in the 5T33MM murine model. We report that the predominant population of CD138+ cells had higher engraftment potential, higher clonogenic growth, whereas the CD138- MM cells presented with less mature phenotype and higher drug resistance. Our findings suggest that while designing treatment regimes for MM, both the cellpopulations must be targeted. In paper IV we have studied the general mechanism of differential gene expression regulation by CGGBP1 in response to growth signals in normal human fibroblasts. We found that CGGBP1 binding affects global gene expression by RNA Polymerase II. This is mediated by Alu RNAdependentinhibition of RNA Polymerase II. In presence of growth signals CGGBP1 is retained in the nuclei and exhibits enhanced Alu binding thus inhibiting RNA Polymerase III binding on Alus. Hence we suggest a mechanism by which CGGBP1 orchestrates Alu RNA-mediated regulation of RNA Polymerase II. This thesis provides new insights for using integrative bioinformatic approaches to decipher gene expression regulation mechanisms in MM and in normal cells.

Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-activated protein present on endothelial cells (ECs). Our laboratory has provided evidence that DNA methylation, a mark associated with gene silencing, is fundamental for regulating VCAM-1 expression. First, we showed that RNA polymerase II, preferentially associates with VCAM-1 hypomethylated alleles. This finding was confirmed using fluorescence-activated cell sorting (FACS) to sort populations of cytokine-activated ECs with high vs. low cell surface VCAM-1 expression. We found that ECs with high VCAM-1 expression were hypomethylated at the promoter. We then went on to show that populations of cells generated from single ECs exhibit differential VCAM-1 methylation from one another, and from the original founder population. Intriguingly, our data shows that VCAM-1 mRNA levels differ between the clones, and correlate with the observed differences in DNA methylation. Taken together, this data provides exciting evidence that DNA methylation is important in the regulation of VCAM-1 gene expression.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-activated protein present on endothelial cells (ECs). Our laboratory has provided evidence that DNA methylation, a mark associated with gene silencing, is fundamental for regulating VCAM-1 expression. First, we showed that RNA polymerase II, preferentially associates with VCAM-1 hypomethylated alleles. This finding was confirmed using fluorescence-activated cell sorting (FACS) to sort populations of cytokine-activated ECs with high vs. low cell surface VCAM-1 expression. We found that ECs with high VCAM-1 expression were hypomethylated at the promoter. We then went on to show that populations of cells generated from single ECs exhibit differential VCAM-1 methylation from one another, and from the original founder population. Intriguingly, our data shows that VCAM-1 mRNA levels differ between the clones, and correlate with the observed differences in DNA methylation. Taken together, this data provides exciting evidence that DNA methylation is important in the regulation of VCAM-1 gene expression.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

Accumulated genetic and epigenetic abnormalities contribute to the development of colon cancer. We have shown that a combination of fish oil (containing decosahexaenoic acid, DHA, 22:6 n-3) and pectin (fermented to butyrate by colonic microflora) is protective against colon carcinogenesis in part by regulating the expression of genes involved in apoptosis, leading to apoptosis induction. To determine how FO/P enhances apoptosis, we measured the expression of genes involved in apoptosis. We performed a pathway analysis on differentially expressed genes identified at three times during colon tumorigenesis: initiation, aberrant crypt foci (ACF) formation, and tumor stage, and compared these results with phenotypic observations at those times. At initiation, FO/P down-regulated the expression of genes involved with cell adhesion and enhanced apoptosis compared with corn oil/cellulose (CO/C). At the ACF stage, expression of genes involved in cell cycle regulation was modulated by FO/P and proliferation was reduced in FO/P rats compared with CO/C rats. FO/P increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We next determined if changes in expression of genes involved in apoptosis by FO/P are associated with changes in promoter methylation of a key apoptosis regulator, Bcl-2. Genomic DNA was isolated from carcinogen-induced colon tumors and non-involved tissues. FO/P increased Bcl-2 promoter methylation in tumor tissues and colonocyte apoptosis relative to those observed with CO/C. A negative correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. Additionally, we examined gene specific promoter methylation of 24 apoptosis-related genes using human colon cancer cells. Cells were treated with DHA or linoleic acid (18:2 n-6), and select cultures were also treated with butyrate. The combination of DHA and butyrate led to a significant reduction in the methylation of pro-apoptotic genes and an increase in apoptosis. These data suggest that part of the mechanisms involved in the induction of apoptosis by FO/P may be through epigenetic regulation of genes involved in apoptosis throughout colon carcinogenesis.

Plants are stationary and need to adapt to the environment they live in. Integration of environmental cues, such as changes in light and temperature, can occur either directly or through the action of hormones. Hormone and light signaling leads to rapid changes in gene expression, and eventually changes in protein levels. In this thesis I have studied how the epigenetic regulator TERMINAL FLOWER2 (TFL2) is involved in light and hormonal signaling in the model organism Arabidopsis thaliana (thale cress). TFL2 is the only Arabidopsis homologue of HETEROCHROMATIN PROTEIN1 (HP1). HP1 proteins have been shown to be involved in repressing gene expression by maintaining the tight structure of heterochromatin or by forming a heterochromatin like structure in euchromatic regions. Unlike metazoan HP1 which can be localized both to eu- and heterochromatin, TFL2 is uniquely localized to euchromatin. tfl2 mutants have reduced levels of free auxin and a reduced rate of auxin biosynthesis. TFL2 binds to and promotes spatial and temporal expression of the genes belonging to the YUCCA gene family, which are believed to regulate a rate limiting step in the auxin biosynthesis pathway. Further, TFL2 binds to a subset of Aux/IAA proteins to repress auxin regulated genes involved in ovule and carpel development. In a similar way, TFL2 is also involved in repressing two jasmonate responsive genes, VEGETATIVE STORAGE PROTEIN1 and 2. This TFL2 regulated repression might occur through the interaction with the jasmonate responsive protein JAZ6. In light signaling TFL2 is involved in repressing both phytochrome A and B signaling as the response to red and far red light is enhanced in tfl2 mutants. The shade avoidance response and chloroplast biogenesis are also regulated by TFL2 as the hypocotyls of tfl2 are not able to elongate as wt in shade conditions and greening is delayed upon de-etiolation of tfl2 seedlings. This work shows that TFL2 has a repressive function in auxin, jasmonate and light signaling and for the first time we show that TFL2 is directly involved in promoting gene expression.

There are two chief molecular pathways to sporadic colorectal cancer (CRC), the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. A third pathway, the pure microsatellite instability pathway, is important in inherited CRC specifically hereditary non-polyposis colorectal cancer. The CIN pathway is characterized by an adenomatous pathological precursor, aneuploidy and microsatellite stability. CIMP pathway cancers, however, are frequently proximal, develop from serrated rather than adenomatous polyps and are strongly associated with BRAF mutation. The CIMP pathway is driven primarily by epigenetic rather than genetic instability. These pathway-specific molecular traits are evident within the pathological precursors to these cancers and thus pathway divergence must occur at the beginning of carcinogenesis or even before. Although DNA methylation is recognized as a key mechanism in colorectal carcinogenesis, relatively little is known about its pattern, regulation and relevance in normal colorectal mucosa. This PhD thesis characterized the profile of DNA methylation in the normal human colorectum and explored its associations with luminal, environmental, dietary and pathological factors. The genes methylated in CRC are characterized as “type A” (Age-related) genes and “type C” (Cancer-specific) genes. Generally, “type A” genes are methylated in both normal and neoplastic tissue with the degree of methylation proportional to the age of the tissue. The methylation of “type C” genes, however, is more specific for neoplastic tissue. The primary study recruited 166 patients undergoing colonoscopy. At colonoscopy, mucosal biopsies were taken from the caecum, transverse colon, sigmoid colon and rectum. DNA methylation was analysed by MethyLight at “type A” (ESR1, GATA5, HIC1, HPP1, SFRP1) and “type C” methylation markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3). LINE-1 methylation was quantified by pyrosequencing. The last 5 “type C” markers comprise a CIMP panel used to identify CIMP cancers. Mean “type A” and CIMP panel methylation Z-scores were calculated. The PMR for each of these CpG island loci was compared to patient age, gender, previous colorectal polyps, smoking history and the presence of concomitant pathology. Most “type A” genes demonstrated strong and direct correlations between methylation and patient age (e.g. ESR1, ρ=0.66, p<0.0001) and had greater methylation within the distal compared to the proximal colorectum (e.g. ESR1, p<0.0001). On multivariate analysis, the mucosal “type A” methylation Z-score had a strong, independent, inverse association with the diagnosis of colorectal adenomas (OR=0.23, p<0.001), the precursor to CIN cancers. The mean CIMP methylation Z-score in normal mucosa, however, was significantly and independently associated with advanced proximal serrated polyps (OR=5.1, p=0.009), the precursor to CIMP cancers. The luminal and epithelial associations with colorectal methylation were explored by a randomized, double-blind, placebo-controlled trial. This experiment was undertaken to determine whether dietary supplementation could modulate epithelial DNA methylation. In addition, the study was designed to evaluate intra-individual reproducibility of the MethyLight technique. The study consisted of a 4 week cross-over trial of resistant starch and Bifidobacterium lactis either alone or as a combined synbiotic preparation, in 20 human volunteers. Rectal biopsies, faeces and serum were collected. Rectal mucosal endpoints included DNA methylation at the CpG island loci and LINE-1, epithelial proliferation (Ki67 immunohistochemistry) and crypt cellularity. Faecal short-chain fatty acid concentrations, pH, ammonia and microbiological profiles (by DGGE and sequencing) were examined. The synbiotic intervention fostered a significantly different faecal stream bacterial community than either the prebiotic or the probiotic interventions alone, but did not show any significant associations with the epithelial or luminal parameters. To explore possible associations between luminal and epithelial parameters and mucosal DNA methylation, the baseline indices were further analysed. There was a strong positive correlation between baseline epithelial proliferation and “type A” marker methylation (ρ = 0.7, p = 0.0001). Thus, “type A” methylation may reflect the cellular age or mitotic burden of a tissue, which is a function of both time and cell turnover. There were consistent inverse trends evident between faecal short-chain fatty acid levels and rectal mucosal DNA methylation. This PhD project found that DNA methylation within the normal colorectal mucosa varied with patient age and region and was strongly associated with the development of pathway-specific pathology, suggesting that the background colorectal field may predict both the at-risk patients and at-risk pathways. Diet and the luminal environment more broadly may influence levels of DNA methylation in the colorectal mucosa and could help to explain regional patterns of colorectal DNA methylation.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 182-187) Also available in print.